GAL1-SceI directed site-specific genomic (gsSSG) mutagenesis: a method for precisely targeting point mutations in S. cerevisiae

Abstract

BackgroundPrecise targeted mutations are defined as targeted mutations that do not require the retention of other genetic changes, such as marker genes, near the mutation site. In the yeast, S. cerevisiae, there are several methods for introducing precise targeted mutations, all of which depend on inserting both a counter-selectable marker and DNA bearing the mutation. For example, the marker can first be inserted, and then replaced with either a long oligonucleotide carrying the mutation (delitto perfetto) or a PCR fragment synthesized with one primer containing the mutation (SSG mutagenesis).ResultsA hybrid method for targeting precise mutation into the genomes uses PCR fragments as in SSG mutagenesis together with a CORE cassette devised for delitto perfetto that contains the homing endonuclease SceI. This method, termed gsSSG mutagenesis, is much more efficient than standard SSG mutagenesis, allowing replacements to be identified without extensive screening of isolates. In gsSSG, recombination between the PCR fragment and the genome occurs equally efficiently regardless of the size of the fragment or the distance between the fragment end and the site of marker insertion. In contrast, the efficiency of incorporating targeted mutations by this method increases as the distance between the mutation and the marker insertion site decreases.ConclusiongsSSG is an efficient way of introducing precise mutations into the genome of S. cerevisiae. The frequency of incorporating the targeted mutation remains efficient at least as far as 460 bp from the insertion site meaning that a single insertion can be used to create many different mutants. The overall efficiency of gsSSG can be estimated based on the distance between the mutation and the marker insertion, and this efficiency can be maximized by limiting the number of untargeted mutations. Thus, a single insertion of marker genes plus homing endonuclease cassette can be used to efficiently introduce precise point mutations through a region of > 900 bp.

Homing endonucleases: DNA scissors on a mission

“…This induces the DSB repair process, which increases the frequency of targeted HR by 4000-fold with engineered DNA introduced by transformation when compared with experiments where DSBs were not generated . Other strategies involving genetic cassettes have been developed that involve I-SceI that allow for targeting point mutations in yeast (Piccirillo et al 2011). The application of re-engineered HEs in plant biotechnology is also gaining interest for developing transformation vector systems for plant genome editing and for targeted mutagenesis (Yang et al 2009;Gao et al 2010;Vainstein et al 2011;Lyznik et al 2012).…”

Size Of Gene Specific Inverted Repeat - Dependent Gene Deletion In Saccharomyces cerevisiae

“…By using the appropriate counter-selectable markers, simple preparation of two or more disruption gene cassettes by OE-PCR enables the simultaneous gene deletion. As with other reported procedures for gene deletions [33], [34], this protocol can be readily adjusted to produce sequence insertions and replacements in the genome. In addition, the higher efficiencies of precise marker excision with the gene-specific inverted sequences demonstrated here can also be easily adapted to other microorganisms for efficient gene deletion.…”